Skip to main content
PMC home page
Protein Science : A Publication of the Protein Society logoLink to Protein Science : A Publication of the Protein Society
. 1993 May;2(5):826–837. doi: 10.1002/pro.5560020512

Electrostatic stabilization in four-helix bundle proteins.

C R Robinson 1, S G Sligar 1
PMCID: PMC2142490  PMID: 8388289

Abstract

Charge substitutions generated by site-directed mutagenesis at the termini of adjacent anti-parallel alpha-helices in a four-helix bundle protein were used to determine a precise value for the contribution of indirect charge-charge interactions to overall protein stability, and to simulate the electrostatic effects of alpha-helix macrodipoles. Thermodynamic double mutant cycles were constructed to measure the interaction energy between such charges on adjacent anti-parallel helices in the four-helix bundle cytochrome b562 from Escherichia coli. Previously, theoretical calculations of helix macrodipole interactions using modeled four-helix bundle proteins have predicted values ranging over an order of magnitude from 0.2 to 2.5 kcal/mol. Our system represents the first experimental evidence for electrostatic interactions such as those between partial charges due to helix macrodipole charges. At the positions mutated, we have measured a favorable interaction energy of 0.6 kcal/mol between opposite charges simulating an anti-parallel helix pair. Pairs of negative or positive charges simulating a parallel orientation of helices produce an unfavorable interaction of similar magnitude. The interaction energies show a strong dependence upon ionic strength, consistent with an electrostatic effect. Indirect electrostatic contacts do appear to confer a limited stabilization upon the association of anti-parallel packing of helices, favoring this orientation by as much as 1 kcal/mol at 20 mM K phosphate.

Full Text

The Full Text of this article is available as a PDF (3.6 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Becktel W. J., Schellman J. A. Protein stability curves. Biopolymers. 1987 Nov;26(11):1859–1877. doi: 10.1002/bip.360261104. [DOI] [PubMed] [Google Scholar]
  2. Bell J. A., Becktel W. J., Sauer U., Baase W. A., Matthews B. W. Dissection of helix capping in T4 lysozyme by structural and thermodynamic analysis of six amino acid substitutions at Thr 59. Biochemistry. 1992 Apr 14;31(14):3590–3596. doi: 10.1021/bi00129a006. [DOI] [PubMed] [Google Scholar]
  3. Carlacci L., Chou K. C. Energetic approach to the folding of four alpha-helices connected sequentially. Protein Eng. 1990 May;3(6):509–514. doi: 10.1093/protein/3.6.509. [DOI] [PubMed] [Google Scholar]
  4. Chou K. C., Maggiora G. M., Némethy G., Scheraga H. A. Energetics of the structure of the four-alpha-helix bundle in proteins. Proc Natl Acad Sci U S A. 1988 Jun;85(12):4295–4299. doi: 10.1073/pnas.85.12.4295. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. DeGrado W. F., Wasserman Z. R., Lear J. D. Protein design, a minimalist approach. Science. 1989 Feb 3;243(4891):622–628. doi: 10.1126/science.2464850. [DOI] [PubMed] [Google Scholar]
  6. Feng Y. Q., Sligar S. G. Effect of heme binding on the structure and stability of Escherichia coli apocytochrome b562. Biochemistry. 1991 Oct 22;30(42):10150–10155. doi: 10.1021/bi00106a011. [DOI] [PubMed] [Google Scholar]
  7. Feng Y. Q., Wand A. J., Sligar S. G. 1H and 15N NMR resonance assignments and preliminary structural characterization of Escherichia coli apocytochrome b562. Biochemistry. 1991 Aug 6;30(31):7711–7717. doi: 10.1021/bi00245a007. [DOI] [PubMed] [Google Scholar]
  8. Fisher M. T. Differences in thermal stability between reduced and oxidized cytochrome b562 from Escherichia coli. Biochemistry. 1991 Oct 15;30(41):10012–10018. doi: 10.1021/bi00105a028. [DOI] [PubMed] [Google Scholar]
  9. Gilson M. K., Honig B. Destabilization of an alpha-helix-bundle protein by helix dipoles. Proc Natl Acad Sci U S A. 1989 Mar;86(5):1524–1528. doi: 10.1073/pnas.86.5.1524. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Goto Y., Hagihara Y. Mechanism of the conformational transition of melittin. Biochemistry. 1992 Jan 28;31(3):732–738. doi: 10.1021/bi00118a014. [DOI] [PubMed] [Google Scholar]
  11. Hahn K. W., Klis W. A., Stewart J. M. Design and synthesis of a peptide having chymotrypsin-like esterase activity. Science. 1990 Jun 22;248(4962):1544–1547. doi: 10.1126/science.2360048. [DOI] [PubMed] [Google Scholar]
  12. Hodgson J. Protein design: rules, empiricism, & nature. Biotechnology (N Y) 1990 Dec;8(12):1245–1247. doi: 10.1038/nbt1290-1245. [DOI] [PubMed] [Google Scholar]
  13. Hol W. G., Halie L. M., Sander C. Dipoles of the alpha-helix and beta-sheet: their role in protein folding. Nature. 1981 Dec 10;294(5841):532–536. doi: 10.1038/294532a0. [DOI] [PubMed] [Google Scholar]
  14. Hol W. G. The role of the alpha-helix dipole in protein function and structure. Prog Biophys Mol Biol. 1985;45(3):149–195. doi: 10.1016/0079-6107(85)90001-x. [DOI] [PubMed] [Google Scholar]
  15. Hol W. G., van Duijnen P. T., Berendsen H. J. The alpha-helix dipole and the properties of proteins. Nature. 1978 Jun 8;273(5662):443–446. doi: 10.1038/273443a0. [DOI] [PubMed] [Google Scholar]
  16. Itagaki E., Hager L. P. Studies on cytochrome b-562 of Escherichia coli. I. Purification and crystallization of cytochrome b-562. J Biol Chem. 1966 Aug 25;241(16):3687–3695. [PubMed] [Google Scholar]
  17. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  18. Landschulz W. H., Johnson P. F., McKnight S. L. The leucine zipper: a hypothetical structure common to a new class of DNA binding proteins. Science. 1988 Jun 24;240(4860):1759–1764. doi: 10.1126/science.3289117. [DOI] [PubMed] [Google Scholar]
  19. Lederer F., Glatigny A., Bethge P. H., Bellamy H. D., Matthew F. S. Improvement of the 2.5 A resolution model of cytochrome b562 by redetermining the primary structure and using molecular graphics. J Mol Biol. 1981 Jun 5;148(4):427–448. doi: 10.1016/0022-2836(81)90185-6. [DOI] [PubMed] [Google Scholar]
  20. Mathews F. S., Bethge P. H., Czerwinski E. W. The structure of cytochrome b562 from Escherichia coli at 2.5 A resolution. J Biol Chem. 1979 Mar 10;254(5):1699–1706. [PubMed] [Google Scholar]
  21. Nicholson H., Becktel W. J., Matthews B. W. Enhanced protein thermostability from designed mutations that interact with alpha-helix dipoles. Nature. 1988 Dec 15;336(6200):651–656. doi: 10.1038/336651a0. [DOI] [PubMed] [Google Scholar]
  22. Nikkila H., Gennis R. B., Sligar S. G. Cloning and expression of the gene encoding the soluble cytochrome b562 of Escherichia coli. Eur J Biochem. 1991 Dec 5;202(2):309–313. doi: 10.1111/j.1432-1033.1991.tb16377.x. [DOI] [PubMed] [Google Scholar]
  23. O'Neil K. T., DeGrado W. F. A thermodynamic scale for the helix-forming tendencies of the commonly occurring amino acids. Science. 1990 Nov 2;250(4981):646–651. doi: 10.1126/science.2237415. [DOI] [PubMed] [Google Scholar]
  24. O'Shea E. K., Klemm J. D., Kim P. S., Alber T. X-ray structure of the GCN4 leucine zipper, a two-stranded, parallel coiled coil. Science. 1991 Oct 25;254(5031):539–544. doi: 10.1126/science.1948029. [DOI] [PubMed] [Google Scholar]
  25. Pace C. N. Measuring and increasing protein stability. Trends Biotechnol. 1990 Apr;8(4):93–98. doi: 10.1016/0167-7799(90)90146-o. [DOI] [PubMed] [Google Scholar]
  26. Pflugrath J. W., Quiocho F. A. Sulphate sequestered in the sulphate-binding protein of Salmonella typhimurium is bound solely by hydrogen bonds. Nature. 1985 Mar 21;314(6008):257–260. doi: 10.1038/314257a0. [DOI] [PubMed] [Google Scholar]
  27. Regan L., DeGrado W. F. Characterization of a helical protein designed from first principles. Science. 1988 Aug 19;241(4868):976–978. doi: 10.1126/science.3043666. [DOI] [PubMed] [Google Scholar]
  28. Richardson J. S., Richardson D. C. Amino acid preferences for specific locations at the ends of alpha helices. Science. 1988 Jun 17;240(4859):1648–1652. doi: 10.1126/science.3381086. [DOI] [PubMed] [Google Scholar]
  29. Rodgers K. K., Pochapsky T. C., Sligar S. G. Probing the mechanisms of macromolecular recognition: the cytochrome b5-cytochrome c complex. Science. 1988 Jun 17;240(4859):1657–1659. doi: 10.1126/science.2837825. [DOI] [PubMed] [Google Scholar]
  30. Rogers N. K., Sternberg M. J. Electrostatic interactions in globular proteins. Different dielectric models applied to the packing of alpha-helices. J Mol Biol. 1984 Apr 15;174(3):527–542. doi: 10.1016/0022-2836(84)90334-6. [DOI] [PubMed] [Google Scholar]
  31. Sali D., Bycroft M., Fersht A. R. Stabilization of protein structure by interaction of alpha-helix dipole with a charged side chain. Nature. 1988 Oct 20;335(6192):740–743. doi: 10.1038/335740a0. [DOI] [PubMed] [Google Scholar]
  32. Serrano L., Fersht A. R. Capping and alpha-helix stability. Nature. 1989 Nov 16;342(6247):296–299. doi: 10.1038/342296a0. [DOI] [PubMed] [Google Scholar]
  33. Serrano L., Horovitz A., Avron B., Bycroft M., Fersht A. R. Estimating the contribution of engineered surface electrostatic interactions to protein stability by using double-mutant cycles. Biochemistry. 1990 Oct 9;29(40):9343–9352. doi: 10.1021/bi00492a006. [DOI] [PubMed] [Google Scholar]
  34. Sheridan R. P., Levy R. M., Salemme F. R. alpha-Helix dipole model and electrostatic stabilization of 4-alpha-helical proteins. Proc Natl Acad Sci U S A. 1982 Aug;79(15):4545–4549. doi: 10.1073/pnas.79.15.4545. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Shirley B. A., Stanssens P., Hahn U., Pace C. N. Contribution of hydrogen bonding to the conformational stability of ribonuclease T1. Biochemistry. 1992 Jan 28;31(3):725–732. doi: 10.1021/bi00118a013. [DOI] [PubMed] [Google Scholar]
  36. Shoemaker K. R., Kim P. S., York E. J., Stewart J. M., Baldwin R. L. Tests of the helix dipole model for stabilization of alpha-helices. Nature. 1987 Apr 9;326(6113):563–567. doi: 10.1038/326563a0. [DOI] [PubMed] [Google Scholar]
  37. Sun D. P., Sauer U., Nicholson H., Matthews B. W. Contributions of engineered surface salt bridges to the stability of T4 lysozyme determined by directed mutagenesis. Biochemistry. 1991 Jul 23;30(29):7142–7153. doi: 10.1021/bi00243a015. [DOI] [PubMed] [Google Scholar]
  38. Warshel A., Aqvist J. Electrostatic energy and macromolecular function. Annu Rev Biophys Biophys Chem. 1991;20:267–298. doi: 10.1146/annurev.bb.20.060191.001411. [DOI] [PubMed] [Google Scholar]
  39. de Vos A. M., Ultsch M., Kossiakoff A. A. Human growth hormone and extracellular domain of its receptor: crystal structure of the complex. Science. 1992 Jan 17;255(5042):306–312. doi: 10.1126/science.1549776. [DOI] [PubMed] [Google Scholar]

Articles from Protein Science : A Publication of the Protein Society are provided here courtesy of The Protein Society

RESOURCES